Raman spectroscopy is concerned with the phenomenon of a frequency change when photons of electromagnetic radiation are inelastically scattered by molecules. If the frequency of incident electromagnetic radiation is νo and that of scattered electromagnetic radiation is νr, then the magnitude of the frequency shift or Raman shift νo−νr=Δν is referred to as Raman frequency. The Raman process can be understood by assuming incident electromagnetic radiation consists of photons with energy hνo. On collision with molecules, a photon may be elastically scattered without change of energy. This gives rise to the so-called “Rayleigh” scattering signal. In some cases, called inelastic, the collision causes the electromagnetic radiation scattering molecule to undergo a quantum transition from one vibrational level to another. Energy needed to make this transition is either taken from the scattered photon (if vibrational transition is from lower to higher energetic level) or transferred to the photon (if vibrational transition is from higher to lover energetic level). As a result, the energy of the scattered photon is different from that it initially possessed. Since, under normal conditions of temperature and pressure, the majority of molecules are in a non-excited state, the probability that a photon will transfer its energy to excite the molecule is greater than the probability of the photon gaining energy as a result of molecular relaxation. The change in the energy of the photon leads to proportional shift of its frequency. Hence, the frequency of scattered photon can be shifted up or down by some value Δν from initial frequency νo. Frequency shift caused by interaction of photon with molecule, resulting in change of the vibrational energy of the molecule is referred to as Raman shift.
The amount of energy that can be taken or transferred from a photon to a molecule during the Raman scattering process is equal to the energy needed to change the state of the molecule from one vibration mode to another. The number of modes in a given molecule is limited and the energy needed for transition from one mode to another is well defined. The number of modes and the transition energy between the modes depends on the structure of the molecule, that is to say, the kind and number of atoms in the molecule, their relative position within the molecule, the kind of bonds between them and so on. As a result, each molecule has a specific pattern of possible transition energies. When monochromatic radiation interacts with a large number of such molecules, the pattern of possible energetic transitions is imaged as a pattern of frequency shifts of scattered radiation relative to the frequency of the incident monochromatic radiation. This pattern is called a Raman spectrum and can be obtained by spectrum analysis of the scattered radiation. The analysis of the frequency shift pattern can give information on the kind of molecules involved. Furthermore, since the number of Raman scattered photons is proportional to the intensity of the incident electromagnetic radiation and the number of molecules interacting, the intensity of the scattered electromagnetic radiation can provide information on the concentration of particular species in the specimen. In particular, Raman spectroscopy has demonstrated a wide range of capabilities in the spectral analysis of organic molecules.
Various kinds of spectrum analyzers can be used for this purpose, but recently spectrometers with integrating photodiode or CCD arrays gain importance in the instruments working in the spectral sensitivity ranges of the applied arrays. The Raman scattered radiation is delivered to an entry port (entry slit) of the analyzer/spectrometer. Usually, the capability of the system to register a signal depends on the strength of the signal, which is proportional to the number of photons received at the detector. Thus the strength of the signal depends on the total number of photons available as well as the efficiency of the system to collect these photons and to channel them to the analyzer and detector. The collecting efficiency of any optical system is determined by the optical invariant (or étendue), which is defined as the product of the radiation beam area at its waist and the angular spread-out of the radiation beam which can be accepted and transferred to photodetector. In efficient optical systems, the étendue cannot be larger than the product of the detector area and the solid angle from which it can collect the radiation. For any given detector both these values are predefined and they set a physical limit to the collecting efficiency of the optical system. Once this limit is reached the only way to increase the signal is to increase intensity of the source, which in case of Raman process depends on intensity of delivered excitation, number of molecules involved and efficiency of the process.
There are two distinguishably different Raman processes: stimulated and random (or ordinary) which differ significantly in terms of efficiency. Stimulated Raman scattering can be very efficient but it occurs only when coherent electromagnetic radiation beam of very high power density, produced for example by a laser, coherently interacts with a large number of molecules. The stimulated scattered radiation is well contained in space and can therefore be easily collected, delivered to the spectrum analyzer and detected. Unfortunately, because of the high power density required, this approach can result in damage to live tissues and cannot be routinely used for in-vivo medical diagnosis (see for example U.S. Pat. No. 5,553,616).
Random Raman scattering takes place when molecules interact with non-coherent or coherent electromagnetic radiation of power density insufficient to produce the stimulated Raman effect. Its efficiency is determined by the probability of inelastic scatter of a single photon on a single molecule. This probability is very small and drops dramatically with increasing wavelength of the applied exciting radiation (energy decrease of incident photons). For this and other technical reasons, radiation with wavelengths, which corresponds to the far infrared is seldom applied for Raman excitation. Unfortunately, because of a competing fluorescence effect, application of radiation from the visible and UV ranges is also undesirable.
The probability of random Raman process is very low and Raman scattered electromagnetic radiation is distributed uniformly in space (there is no preferred direction). Furthermore, only a small part of the radiation can be collected due to limited capabilities of radiation collecting systems. As a result, the collected Raman signal is very weak and a lot of effort has been undertaken to increase the collecting efficiency of the applied optical system by increasing the collecting angle as much as possible. Unfortunately, this strategy has not met with much success for two main reasons. The first is that there is an absolute limit, to which the collecting angle can be increased. This collecting angle can not be larger than the full solid angle. In practice the collecting angle is usually many times smaller because of technical limitations. The second is that an increase in the collecting angle reduces the area and volume of the sample from which the scattered radiation can be efficiently collected. This results in reducing the number of molecules being in a field of view of a collecting system, and reduces the number of molecules from which the Raman scattered radiation can be efficiently collected. In this situation, illumination with exciting radiation of the sample area that is larger than that, from which the scattered radiation can be effectively collected, is wasteful and should not be applied. Therefore the option of increasing the signal through improvement of the collecting capability of the optical system is very limited. Application of non-imaging optical systems, which usually collect radiation from larger volumes, is less efficient, and does not solve the problem of weak signal obtained from Raman scattering. Therefore, other methods to increase signal are required.
One way to increase the signal is to increase the intensity of the exciting radiation in the sample volume, from which scattered radiation can be efficiently collected. Unfortunately, many samples, especially of organic origin, have a limited resistance to irradiance with electromagnetic radiation, and there exists a limit for power density beyond which the molecular bonds of the sample can be irreversibly damaged. Therefore, the product of the volume, from which radiation can be efficiently collected, and the maximum power density tolerated by the sample determines a maximum power, which can be reasonably applied for a given sample. Application of a radiation beam with larger power will not produce any gain, either due to the sample damage, or inefficient sample excitation. Because of low efficiency of the random Raman process, direct excitation is not very efficient from energetic point of view.
Another way to increase efficiency is to enforce multiple interaction of radiation with the sample. This idea is exploited in U.S. Pat. No. 4,645,340 which discloses the use of an internally reflective sphere to redirect an unused part of the radiation back to a centrally located sample. The sphere is purely reflective, and no scattering of the radiation by the sphere takes place.
U.S. Pat. No. 4,127,329 teaches a method to increase efficiency of the Raman process, using two or more spherical mirrors to multiply reflect excitation to a gaseous sample. There is no scattering of the radiation by the apparatus.
U.S. Pat. No. 5,506,678 discloses the use of a reflecting tube and a radiation collecting optical system. The radiation signal is collected and delivered to a spectrometer, following interaction between the radiation and a gas sample placed within the tube. There is no scattering of the introduced radiation by the reflecting tube itself.
Collectively, these references address the problem of increasing efficiency of the Raman process through multiple reflection and refocusing of exciting radiation by means of mirrors, and collection of the radiation with radiation collecting optics that are able to collect radiation from a limited volume. Because of the limited volume from which radiation can be efficiently collected, great care is required to increase the power density of exciting radiation in the volume and to increase the poser density in such a way to enhance the probability of Raman scattering process. Because of a limited number of molecules involved in Raman scattering, increasing the power density creates a danger of sample damage. If the samples demonstrate strong elastic scattering, then this approach, is applicable to very small samples only. None of the above identified publications suggests application of an integrating cavity to redirect scattered radiation to a sample, and to store radiation inside the cavity, until it finds a way out of the cavity through one of several possible ports, where one or more of the ports could be coupled with spectrum analyzer and detector.
In WO 97/23159 an integrating cavity is disclosed that is used for spectroscopic measurement. Broad band spectroscopic radiation of known spectral content is introduced into the integrating cavity containing a test sample, and the spectrum of radiation is modified due to absorption in the sample. The spectrally modified radiation is collected and subjected to analysis to obtain information on absorbance of the sample. This publication does not teach application of the integrating cavity for analysis of Raman scattered radiation.
In many applications, for example, the analysis of living human subjects, it is impractical to take a small sample and to place it in the center of a sphere. Furthermore, very often it is important to have a signal from as large an area, or volume, of the sample as possible to reduce the dependence on local variations in the properties of the sample. Due to limited resistance of some samples to high optical power, the signal, which can be obtained from a small sample, is often too weak to provide required information, and application of increased total power is therefore desired. Additionally, in some samples it is important to get the signal from deeper layers of the sample. None of these problems can be addressed with instrumentation presently used for Raman signal collection.
The present invention offers an alternative way for permitting the use of higher power electromagnetic radiation and for the efficient collection of Raman scattered electromagnetic radiation, and overcomes limitations of the prior art.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.